The long term goal of this research is to determine the molecular basis of membrane traffic in mammalian cells. The focus is on mannose 6-phosphate receptors (MPRs) that deliver newly synthesized lysosomal enzymes from the Golgi to pre-lysosomes, and then return to the Golgi to pick up more cargo. We have shown that the protein, GCC185 is needed for tethering of MPR-containing vesicles at the trans Golgi. To verify how GCC185 is anchored and to define the molecular composition of the microdomain where GCC185 functions on the Golgi surface, we will use a novel biotinylation approach to tag nearest neighbors. This will help us understand the difference between the distinct domains occupied by GC185 and Golgi-245 on the trans Golgi. To investigate the mechanism of MPR vesicle tethering at the Golgi, we will analyze the structure of GCC185 using atomic force microscopy and tracking fluorescence correlation spectroscopy, to analyze the importance of core flexibility for tethering function. We will also carry out rescue experiments to test the function of mutant proteins that may not be flexible. Our structural data suggest that the protein may collapse on the Golgi rather than bend in the middle. To distinguish directly between tether bending and tether collapse, we will attempt to biotinylate only GCC185 peptides that are in close proximity to the Golgi surface and map these onto the GCC185 protein sequence. If GCC185 lies down on the Golgi, all peptides should be biotinylatable. Finally, we will isolate fluorescently labeled, mannose 6-phosphate receptor-containing vesicles and monitor how these engage the GCC185 tether. Where do the vesicles bind? What models best explain how GCC185 tethers vesicles at the Golgi? In summary, these experiments open up entirely new areas of investigation in the area of MPR trafficking and will provide fundamental information regarding the mechanisms of vesicle tethering at the Golgi in human cells. The work has broad application to our understanding of a number of disease states including diabetes, cancer, heart disease and neurological disorders.